Project Summary Treatment of immunological disorders with emerging treatments such as immunotherapy represent a growing medical paradigm. To create potent and targeted therapies, defining the underlying immunological mechanisms of action and is critical. Because of immunological complexity, measurements with single cell resolution are essential as both diseases and treatments are often driven by small subpopulations of cells. However, due to limitations with current cell isolation and analysis tools, precisely mapping these complex interactions with single cell resolution is difficult and cost prohibitive to deploy at a scale conducive for high throughput target discovery or streamlined clinical monitoring. Most high precision single cell isolation and analysis tools, such as FACS or microfluidic platforms, are too low throughput and are difficult to integrate into SBS formats for high throughput screening (HTS) workflows. Contrastingly, bulk cell sorting methods such as MACS are easily integrated into HTS formats but cannot achieve single cell capture or analysis. The inherent tradeoff of single cell precision and scalability impeads immunological R&D and clinical translation efforts. Through primary market research interviews using I Corp techniques, researchers have voiced their dissatisfaction with the current single cell isolation tools saying they are a ?major roadblock in our work on phenotypic analysis.? We reasoned that a product that can integrate cell isolation and single cell analysis in an SBS well plate format would alleviate these bottlenecks and accelerate clinical translation. Leveraging a core technology known as ratcheting cytometry, the objective of this proposal to develop a ?smart plate? which can achieve integrated cell purification and arrangement into individual cells for single cell interrogation in SBS format. In preliminary studies, ratcheting cytometry has demonstrated quantitative cell sorting capacity and capacity to self-assemble cells into a single cell format. In phase I, ratcheting cytometry chips will be used to (1) purify CD 14+ monocytes into individual cells followed by (2) co-localization of commercially available magnetic ELISA beads to measure TNF ? secretion activity of single cells. Phase II will focus on scaling the ratcheting cytometry cartridges and integration into SBS well plate format for HTS workflows.